Search

BackgroundWithin eight weeks of primary Clostridioides difficile infection (CDI), as many as 30% of patients develop recurrent disease with the associated risks of multiple relapses, morbidity, and economic burden. There are no validated biomarkers predictive of recurrence during primary infection. This study demonstrates the potential of a simple test for identifying hospitalized CDI patients at low risk for disease recurrence.MethodsForty-six hospitalized CDI patients were enrolled at Emory University Hospitals. Serum and MENSA samples prepared during weeks 1, 2, and 4 following symptom-onset were measured for antibodies specific for ten C. difficile antigens.ResultsAmong the 46 C. difficile-infected patients, nine (19.5%) experienced recurrence within eight weeks of primary infection. Among the 37 non-recurrent patients, 23 had anti-C. difficile MENSA antibodies specific for any of the three toxin antigens: TcdB-CROP, TcdBvir-CROP, and/or CDTb. Positive MENSA responses occurred within the first week post-symptom onset, including six patients who never seroconverted. A similar trend was observed in serum responses, but they peaked later and identified fewer patients (19/37). In contrast, none of the patients who subsequently recurred after hospitalization produced antibodies specific for the three C. difficile toxin antigens. IgA antibodies for the toxin antigens demonstrated the greatest predictive power for protection from recurrence.DiscussionThe development of IgG and/or IgA antibodies for three C. difficile toxins in serum and/or MENSA has prognostic potential. These immunoassays measure nascent immune responses that reduce the likelihood of recurrence. Early identification of patients at-risk for recurrence can reduce costs and morbidity.

It is unclear how the brain network changed after kidney transplantation (KT). We explored the patterns of large‐scale complex network after KT in end‐stage renal disease (ESRD) patients with resting‐state functional MRI (rs‐fMRI) and diffusion tensor imaging (DTI). Twenty‐one ESRD patients (14 men; mean age, 31.5 ± 9.9 years) scheduled for KT and 17 age‐ and gender‐matched healthy controls (HC) (8 men; mean age, 28.9 ± 7.2 years) were enrolled in this study. Each participant underwent rs‐fMRI and DTI scans in three time points (pre‐KT, 1 and 6 months after KT [for ESRD]). Graph theory analysis was used to characterize the topological properties by using functional and structural network connectivities intergroup correlation analysis was performed between functional/structural MR indexes and clinical markers. Compared with HC, pre‐KT ESRD patients showed an altered topological organization in both functional and structural networks. Compared with pre‐KT, increased node degree and node efficiency were observed for both functional and structural networks at 1 month after KT (all p

DNase1L3 deficiency is an inborn error of immunity that causes monogenic systemic lupus erythematosus (SLE) in humans. Here, we identified that one third of patients with sporadic SLE have antibodies to DNase1L3. Like DNase1L3 deficiency, we found that patients with anti-DNase1L3 antibodies have features associated with immune pathways activated by immunogenic self-DNA, including elevated antibodies to dsDNA and prominent expression of the interferon and myeloid/neutrophil signatures. Interestingly, 40-80% of anti-DNase1L3 antibodies in SLE serum contain the 9G4 idiotype, which is encoded by the autoreactive heavy-chain gene segment VH4-34. Sequence and functional analysis of four anti-DNase1L3 monoclonal antibodies generated from SLE patients experiencing disease-associated flares showed that these antibodies were derived from self-reactive 9G4+ switched memory B cells. These antibodies are highly enriched in somatic hypermutations, indicating that they originated from antigen-experienced cells, and have neutralizing activity against DNase1L3. Together, the data demonstrate that autoantibodies to DNase1L3 phenocopy pathogenic mechanisms associated with DNase1L3 deficiency. Moreover, the finding that autoreactive B cells bearing the 9G4 idiotype produce dominant serum autoantibodies, including antibodies to DNase1L3, underscores VH4-34+ B cells as sensible therapeutic targets for specific depletion of pathogenic B cells in SLE.

Background Cryptococcus neoformans is a major cause of morbidity and mortality among human immunodeficiency virus (HIV)-infected persons worldwide, and there are scarce recent data on cryptococcal antigen (CrAg) positivity in the United States We sought to determine the frequency of cryptococcal disease and compare the performance of a CrAg lateral flow assay (LFA) versus latex agglutination (LA) test. Methods All patients from Grady Health System in Atlanta who had a serum or cerebrospinal fluid (CSF) sample sent for CrAg testing as part of clinical care from November 2017 to July 2018 were included. Percentage positivity and test agreement were calculated. Results Among 467 patients, 557 diagnostic tests were performed; 413 on serum and 144 on CSF. The mean age was 44 years, and most were male (69%) and had HIV (79%). Twenty-four (6.4%, 95% confidence interval [CI] = 4.1–9.4) patients were serum CrAg positive, and 8 (5.8%, 95% CI = 2.6–11.2) individuals tested positive for CSF CrAg. Although overall agreement between the LA and LFA was substantial to high for CSF (κ = 0.71, 95% CI = 0.51–0.91) and serum (κ = 0.93, 95% CI = 0.86–1.00), respectively, there were important discrepancies. Five patients had false-positive CSF LA tests that affected clinical care, and 4 patients had discordant serum tests. Conclusions We found a moderately high proportion of cryptococcal disease and important discrepancies between the LA test and LFA. Clinical implications of these findings include accurate detection of serum CrAg and averting unnecessary treatment of meningitis with costly medications associated with high rates of adverse events., We sought to determine the proportion of cryptococcal antigen (CrAg) positivity and compare a CrAg lateral flow assay (LFA) versus latex agglutination test in Atlanta, GA. CrAg positivity was moderately high, and there were important discrepancies between diagnostic tests.

Nasopharyngeal swabs (NPS) collected by trained healthcare professionals are the preferred specimen for SARS-CoV-2 testing. Self-collected specimens might decrease patient discomfort, conserve healthcare resources, and be preferred by patients. During August – November 2020, 1,806 adults undergoing SARS-CoV-2 testing in Denver, Colorado and Atlanta, Georgia, provided self-collected anterior nares swabs (ANS) and saliva specimens before NPS collection. Compared to NPS, sensitivity for SARS-CoV-2 detection by rRT-PCR appeared higher for saliva than for ANS (85% versus 80% in Denver; 67% versus 58% in Atlanta) and higher among participants reporting current symptoms (94% and 87% in Denver; 72% and 62% in Atlanta, for saliva and ANS, respectively) than among those reporting no symptoms (29% and 50% in Denver; 50% and 44% in Atlanta, for saliva and ANS, respectively). Compared to ANS, saliva was more challenging to collect and process. Self-collected saliva and ANS are less sensitive than NPS for SARS-CoV-2 detection; however, they offer practical advantages and might be most useful for currently symptomatic patients.

Background Streptococcus pneumoniae infections cause morbidity and mortality worldwide. A rapid, simple diagnostic method could reduce the time needed to introduce definitive therapy potentially improving patient outcomes. Methods We introduce two new methods for diagnosing S. pneumoniae infections by measuring the presence of newly activated, pathogen-specific, circulating Antibody Secreting Cells (ASC). First, ASC were detected by ELISpot assays that measure cells secreting antibodies specific for signature antigens. Second, the antibodies secreted by isolated ASC were collected in vitro in a novel matrix, MENSA (media enriched with newly synthesized antibodies) and antibodies against S. pneumoniae antigens were measured using Luminex immunoassays. Each assay was evaluated using blood from S. pneumoniae and non-S. pneumoniae-infected adult patients. Results We enrolled 23 patients with culture-confirmed S. pneumoniae infections and 24 controls consisting of 12 non-S. pneumoniae infections, 10 healthy donors and two colonized with S. pneumoniae. By ELISpot assays, twenty-one of 23 infected patients were positive, and all 24 controls were negative. Using MENSA samples, four of five S. pneumoniae-infected patients were positive by Luminex immunoassays while all five non-S. pneumoniae-infected patients were negative. Conclusion Specific antibodies produced by activated ASC may provide a simple diagnostic for ongoing S. pneumoniae infections. This method has the potential to diagnose acute bacterial infections.

BACKGROUNDClostridioides difficile infections (CDI) have been a challenging and increasing serious concern in recent years. While early and accurate diagnosis is crucial, available assays have frustrating limitationsOBJECTIVEDevelop a simple, blood-based immunoassay to accurately diagnose patients suffering from active CDI.MATERIALS AND METHODSUninfected controls (n=95) and CDI patients (n=167) were recruited from Atlanta area hospitals. Blood samples were collected from patients within twelve days of a positive CDI test and processed to yield serum and PBMCs cultured to yield medium enriched for newly synthesized antibodies (MENSA). Multiplex immunoassays measured Ig responses to ten recombinant C. difficile antigens.RESULTSSixty-six percent of CDI patients produced measurable responses to C. difficile antigens in their serum or MENSA within twelve days of a positive CDI test. Fifty-two of the 167 CDI patients (31%) were detectable in both serum and MENSA, but 32/167 (19%) were detectable only in MENSA, and 27/167 (16%) were detectable only in serum.DISCUSSIONWe describe the results of a multiplex immunoassay for the diagnosis of ongoing CDI in hospitalized patients. Our assay resolved patients into four categories: MENSA-positive only, serum-positive only, MENSA- and serum-positive, and MENSA- and serum-negative. The MENSA positive-only patients accounted for 30% and may be attributed to nascent antibody secretion in MENSA prior to seroconversion. Conversely, the serum positive-only subset may have been more advanced in their disease course. Immunocompromise and misdiagnosis may have contributed to the 34% of CDI patients who were not identified using MENSA or serum immunoassays.IMPORTANCEWhile there was considerable overlap between patients identified through MENSA and serum, both methods detected additional, unique patients. The combined use of both MENSA and serum to detect CDI patients resulted in the greatest identification of CDI patients. Together, longitudinal analysis of MENSA and serum will provide a more accurate evaluation of successful host humoral immune responses in CDI patients.

Background Geenius HIV 1/2 Supplemental Assay (Geenius; Bio-Rad Laboratories) is the only Food and Drug Administration-approved HIV-1/HIV-2 antibody differentiation test for the second step in the HIV laboratory testing algorithm. We characterized the occurrence of true HIV-1 and HIV-2 infections as well as false results in 6 US clinical laboratories using Geenius. Methods We examined routine HIV testing outcome data from the time the laboratories began using the algorithm with Geenius until September 30, 2017. We calculated the positive predictive value for Geenius HIV-1 and HIV-2 reactivity separately. Results Of 5,046,684 specimens tested, 41,791 had reactive antigen/antibody test results. Most specimens with reactive antigen/antibody results were HIV-1 antibody-positive established infections (n = 32,421), 1,865 of which also had indeterminate HIV-2 bands present. Ninety-three specimens were HIV-2 antibody positive or untypable for HIV-1/HIV-2 antibody. Acute HIV-1 infections were found in 528 specimens; 881 specimens lacked the nucleic acid test to determine the possibility of acute HIV-1 infection. False-positive antigen/antibody test results were present in 7505 specimens. Few specimens (n = 363) had false-positive antigen/antibody results with indeterminate Geenius and negative HIV-1 nucleic acid test results. The positive predictive values of Geenius reactivity were 99.4% for HIV-1 and 4.3% for HIV-2. Conclusions Routine testing using the laboratory testing algorithm with Geenius resulted in most specimens resolving as HIV negative or HIV-1 positive. The occurrence of indeterminate HIV-2 bands with a Geenius final assay interpretation of HIV-1 positive was more common than true HIV-2 infections. Reporting indeterminate HIV-2 results in this situation may cause confusion with interpreting HIV infection status.

The association between human immunodeficiency virus (HIV) infection and tuberculosis (TB) mortality has been studied extensively, but the impact of HIV on other clinically relevant aspects of TB care such as TB drug-related adverse events (AEs), hospital readmissions, and TB treatment duration is less well characterized. We describe the association of HIV infection with TB clinical complexities and outcomes in a high HIV prevalence cohort in the United States. This is a retrospective cohort study among patients treated for culture-confirmed TB between 2008 and 2015 at an inner-city hospital in Atlanta, GA. Univariate analysis was used to estimate association of HIV with TB treatment interruption due to AEs, hospital readmissions, and treatment duration. Final unfavorable TB treatment outcome was defined as death, loss to follow-up, or recurrent TB. Logistic regression modeling was used to estimate association of HIV with final unfavorable outcomes. Among 274 patients with TB, 96 (35%) had HIV coinfection. HIV-positive patients had more TB treatment interruptions due to AE (34% vs. 15%), were more likely to have a hospital readmission (50% vs. 21%), and received longer TB treatment (9.9 months vs. 8.8 months) compared to HIV-negative patients (p

Campylobacteriosis is an infectious gastrointestinal disease caused by Campylobacter spp. In most cases, it is either underdiagnosed or underreported due to poor diagnostics and limited databases. Several DNA-based molecular diagnostic techniques, including 16S ribosomal RNA (rRNA) sequence typing, have been widely used in the species identification of Campylobacter. Nevertheless, these assays are time-consuming and require a high quality of bacterial DNA. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS is an emerging diagnostic technology that can provide the rapid identification of microorganisms by using their intact cells without extraction or purification. In this study, we analyzed 24 American Type Culture Collection reference isolates of 16 Campylobacter spp. and five unknown clinical bacterial isolates for rapid identification utilizing two commercially available MADI-TOF MS platforms, namely the bioMérieux VITEK® MS and Bruker Biotyper systems. In addition, 16S rRNA sequencing was performed to confirm the species-level identification of the unknown clinical isolates. Both MALDI-TOF MS systems identified the isolates of C. jejuni, C. coli, C. lari, and C. fetus. The results of this study suggest that the MALDI-TOF MS technique can be used in the identification of Campylobacter spp. of public health importance.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative option for treatment of some malignant and non-malignant hematological diseases. However, post-HSCT patients are severely immunocompromised and susceptible to viral infections, which are a major cause of morbidity and mortality. Although antiviral agents are now available for most types of viral infections, they are not devoid of side effects and their efficacy is limited when there is no concomitant antiviral immune reconstitution. In recent decades, adoptive transfer of viral-specific T cells (VSTs) became an alternative treatment for viral infection after HSCT. However, two major issues are concerned in VST transfer: the risk of GVHD and antiviral efficacy. We report an exhaustive review of the published studies that focus on prophylactic and/or curative therapy by donor VST transfer for post-HSCT common viral infections. A low incidence of GVHD and a good antiviral efficacy was observed after adoptive transfer of VSTs from HSCT donor. Viral-specific T-cell transfer is a promising approach for a broad clinical application. Nevertheless, a randomized controlled study in a large cohort of patients comparing antiviral treatment alone to antiviral treatment combined with VSTs is still needed to demonstrate efficacy and safety.

BACKGROUND Children with sickle cell disease (SCD) are at increased risk for invasive infection with encapsulated bacteria. Antibiotic prophylaxis and immunizations against Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) have decreased the overall incidence of invasive infections and have shifted distribution of serotypes causing disease toward those not covered by immunizations. We sought to determine the current incidence of invasive H. influenzae infections in children with SCD and to describe the clinical features and management of these infections. METHODS Microbiology reports of a large pediatric tertiary care center were reviewed to identify all isolates of H. influenzae detected in sterile body fluid cultures from January 1, 2010 to December 31, 2017. Results were compared with the center's comprehensive clinical database of all children with SCD to identify all cases of children ages 0 to18 years with SCD with invasive H. influenzae disease for the same time period. RESULTS We captured 2444 patients with SCD, with 14,336 person-years. There were eight episodes of H. influenzae bacteremia in seven children with SCD (five type f, two non-typable, one type a). Most episodes (7 of 8) were in children

Background Antiretroviral therapy (ART) for persons with HIV infection prevents tuberculosis (TB) disease. Additionally, sequential ART after initiation of TB treatment improves outcomes. We examined ART use, retention in care, and viral suppression (VS) before, during, and 3 years following TB treatment for an inner-city cohort in the United States. Methods Retrospective cohort study among persons treated for culture-confirmed TB between 2008 and 2015 at an inner-city hospital. Results Among 274 persons with culture-confirmed TB, 96 (35%) had HIV co-infection, including 23 (24%) new HIV diagnoses and 73 (76%) previous diagnoses. Among those with known HIV prior to TB, the median time of known HIV was 6 years, and only 10 (14%) were on ART at the time of TB diagnosis. The median CD4 at TB diagnosis was 87 cells/uL. Seventy-four (81%) patients received ART during treatment for TB, and 47 (52%) has VS at the end of TB treatment. Only 32% of patients had continuous VS 3 years after completing TB treatment. There were 3 TB recurrences and 3 deaths post–TB treatment; none of these patients had retention or VS after TB treatment. Conclusions Among persons with active TB co-infected with HIV, we found that the majority had known HIV and were not on ART prior to TB diagnosis, and retention in care and VS post–TB treatment were very low. Strengthening the HIV care continuum is needed to improve HIV outcomes and further reduce rates of active TB/HIV co-infection in our and similar settings.

Background While Cryptococcus neoformans is a major cause of morbidity and mortality among HIV-infected persons worldwide, there is scarce recent data on disease prevalence in the United States, including in Southeastern states, where HIV rates are high. We sought to determine the prevalence of cryptococcal disease and compare the performance of a cryptococcal antigen (CrAg) lateral flow assay (LFA) vs. latex agglutination (LA) test. Methods All patients from Grady Memorial Hospital in Atlanta, Georgia who had a serum or cerebrospinal fluid (CSF) sample sent for CrAg LA testing as part of routine management from November 2017 to July 2018 were included. The LFA was performed on all samples by research staff; results were not available to clinicians. Rates of disease and agreement between the LA test and LFA were calculated. Results Among 467 patients, 570 LA tests were performed; 417 on serum and 153 on CSF (87 patients with multiple tests performed). Mean age was 44 years, and most were male (n = 322, 69%). Most patients had HIV (n = 371, 79%); median CD4 count was 73 cells/mm3 and 77% were not receiving ART. Among HIV-infected individuals, testing was performed equally in the inpatient and outpatient setting. Cryptococcal testing was done in 53 persons without apparent risk factors. Thirty-three (7%) patients had a positive serum or CSF test. Five (1%) patients had both a positive serum and CSF LA test and LFA. While the overall agreement between the LA test and LFA was substantial to high for CSF (κ = 0.71) and serum (κ = 0.93), respectively, there were important discrepancies. Four patients with a negative serum LA test had a positive serum LFA. Five patients had false-positive CSF LA tests, determined by negative CSF LFA testing, India ink, and CSF and fungal cultures. All were treated with amphotericin and flucytosine with one patient experiencing a severe anaphylactic reaction to amphotericin. Conclusion We found a moderately high rate of cryptococcal disease and important discrepancies between the LA test and LFA. The LFA appeared to be more sensitive for cryptococcemia and more specific for meningitis. Clinical implications of these findings include earlier detection and treatment of cryptococcemia, and averting unnecessary treatment of meningitis with costly medications associated with high rates of adverse events. Disclosures All authors: No reported disclosures.

Background Previous studies showed improvement in heart function by injecting bone marrow mesenchymal stem cells (BMSCs) after AMI. Emerging evidence suggested that both the number and function of BMSCs decline with ageing. We designed a randomized, controlled trial to further investigate the safety and efficacy of this treatment. Methods Patients with ST-elevation AMI undergoing successful reperfusion treatment within 12hours were randomly assigned to receive an intracoronary infusion of BMSCs (n=21) or standard medical treatment (n=22) (the numbers of patients were limited because of the complication of coronary artery obstruction). Results There is a closely positive correlation of the number and function of BMSCs vs. the cardiac function reflected by LVEF at baseline (r=0.679, P=0.001) and at 12-month follow-up (r=0.477, P=0.039). Six months after cell administration, myocardial viability within the infarct area by 18-FDG SPECT was improved in both groups compared with baseline, but no significant difference in the BMSCs compared with control groups (4.0±0.4% 95%CI 3.1–4.9 vs. 3.2±0.5% 95%CI 2.1–4.3, P=0.237). 99mTc-sestamibi SPECT demonstrated that myocardial perfusion within the infarct area in the BMSCs did not differ from the control group (4.4±0.5% 95%CI 3.2–5.5 vs. 3.9±0.6% 95%CI 2.6–5.2, P=0.594). Similarly, LVEF after 12 and 24months follow-up did not show any difference between the two groups. In the BMSCs group, one patient suffered a serious complication of coronary artery occlusion during the BMSCs injection procedure. Conclusions The clinical benefits of intracoronary injection of autologous BMSCs in acute STEMI patients need further investigation and reevaluation.

This case report discusses a patient with sickle cell disease who presented with fungemia from Pichia anomala (teleomorph: Candida pelliculosa). The organism was identified as P. anomala by MALDI-TOF VITEK mass spectrometry and VITEK 2 yeast identification card. Pichia anomala should be considered in sickle cell patients with recurrent fungemia.

Both male and female rat growth plate chondrocytes express estrogen receptors (ERs); however 17β-estradiol (E2) induces membrane responses leading to activation of phospholipase A2 (PLA2), phospholipase C (PLC), prostaglandin E2 (PGE2) production, protein kinase C (PKC), and ultimately mitogen protein kinase (MAPK) only in female cells. This study investigated if these sex-specific responses are due to differences in the actual ERs or in downstream signaling. Western blots and flow cytometry of costochondral cartilage resting zone chondrocytes (RCs) showed 2–3 times more ERα in plasma membranes (PMs) from female cells than male cells. Tunicamycin blocked E2-dependent ER-translocation to the PM, indicating palmitoylation was required. Co-immunoprecipitation showed E2 induced complex formation between ER isoforms only in female RCs. To examine if the lack of response in PKC and PGE2 in males is due to differences in signaling, we examined involvement of ERs and the role of PLC and PLA2. Selective ERα (propylpyrazole triol, PPT) and ERβ (diarylproprionitrile, DPN) agonists activated PKC in female RCs only. The PLC inhibitor, U73122 blocked E2's effect on PKC and the cytosolic PLA2 inhibitor, AACOCF3 inhibited the effect on PGE2 in female RCs, confirming involvement of PLC and PLA2 in the mechanism. The PLC activator, m-3M3FβS activated PKC and PLAA peptide increased PGE2 levels in male and female RCs, showing that the signaling pathways are present. These data indicate that differences in membrane ER amount, localization, translocation and interaction are responsible for the sexual dimorphic response to E2.

Multidrug-resistant tuberculosis (MDR TB), defined by resistance to the 2 most effective first-line drugs, isoniazid and rifampin, is on the rise globally and is associated with significant morbidity and mortality. Despite the increasing availability of novel rapid diagnostic tools for Mycobacterium tuberculosis (Mtb) drug susceptibility testing, the clinical applicability of these methods is unsettled. In this study, the mechanisms of action and resistance of Mtb to isoniazid and rifampin, and the utility, advantages and limitations of the available Mtb drug susceptibility testing tools are reviewed, with particular emphasis on molecular methods with rapid turnaround including line probe assays, molecular beacon-based real-time polymerase chain reaction and pyrosequencing. The authors conclude that neither rapid molecular drug testing nor phenotypic methods are perfect in predicting Mtb drug susceptibility and therefore must be interpreted within the clinical context of each patient.

Human papillomavirus (HPV) was found to be the causative agent for cervical cancer in the 1980s with almost 100% of cervical cancer cases testing positive for HPV. Since then, many studies have been conducted to elucidate the molecular basis of HPV, the mechanisms of carcinogenesis of the virus, and the risk factors for HPV infection. Traditionally, the Papanicolaou test was the primary screening method for cervical cancer. Because of the discovery and evolving understanding of the role of HPV in cervical dysplasia, HPV testing has been recommended as a new method for cervical cancer screening by major professional organizations including the American Cancer Society, American Society for Colposcopy and Cervical Pathology, and the American Society for Clinical Pathology. In order to detect HPV infections, many sensitive and specific HPV assays have been developed and used clinically. Different HPV assays with various principles have shown their unique advantages and limitations. In response to a clear causative relationship between high-risk HPV and cervical cancer, HPV vaccines have been developed which utilize virus-like particles to create an antibody response for the prevention of HPV infection. The vaccines have been shown in long-term follow-up studies to be effective for up to 8 years; however, how this may impact screening for vaccinated women remains uncertain. In this chapter, we will review the molecular basis of HPV, its pathogenesis, and the epidemiology of HPV infection and associated cervical cancer, discuss the methods of currently available HPV testing assays as well as recent guidelines for HPV screening, and introduce HPV vaccines as well as their impact on cervical cancer screening and treatments.

Harnessing the differentiation of stem cells into bone-forming cells represents an intriguing avenue for the creation of functional skeletal tissues. Therefore, a profound understanding of bone development and morphogenesis sheds light on the regenerative application of stem cells in orthopedics and dentistry. In this concise review, we summarize the studies deciphering the mechanisms that govern osteoblast differentiation in the context of in vitro formation of bone-like nodules, including morphologic and molecular events as well as cellular contributions to mineral nucleation, occurring during osteogenic differentiation of stem cells. This article also highlights the limitations of current translational applications of stem cells and opportunities to use the bone-like nodule model for bone regenerative therapies. Significance Harnessing the differentiation of stem cells into bone-forming cells represents an intriguing avenue for the creation of functional skeletal tissues. Therefore, a profound understanding of bone development and morphogenesis sheds light on the regenerative application of stem cells in orthopedics and dentistry. In this concise review, studies deciphering the mechanisms that govern osteoblast commitment and differentiation are summarized. This article highlights the limitations of current translational applications of stem cells and the opportunities to use the bone-like nodule model for bone regenerative therapies.

This study is a retrospective chart review looking at the clinical characteristics of cryptococcal meningitis with elevated MIC to fluconazole in immunocompromised patients. These patients were more likely to have central nervous system complications without any effect on mortality., Background. Mortality for cryptococcal meningitis remains significant, in spite of available treatment. Resistance to first-line maintenance therapy, particularly fluconazole, has been reported. Methods. A retrospective chart review was performed on immunocompromised patients with cryptococcal meningitis, who had susceptibility testing performed between January 2001 and December 2011, at 3 hospitals in Atlanta, Georgia. Results. A total of 35 immunocompromised patients with cryptococcal meningitis were identified, 13 (37.1%) of whom had an elevated minimum inhibitory concentration (MIC) to fluconazole (MIC ≥16 µg/mL). Eighty percent of patients were males with African American predominance, the median age was 37 years, and 80% of the patients were human immunodeficiency virus (HIV) positive. Subsequent recurrence of cryptococcal meningitis was more likely in HIV patients compared with solid organ transplant patients (P = .0366). Overall, there was a statistically significant increase in an elevated MIC to fluconazole in patients who had a history of prior azole use (odds ratio, 10.12; 95% confidence interval, 2.04–50.16). Patients with an elevated MIC to fluconazole and those with a high cerebrospinal fluid cryptococcal antigen load (≥1:512) were more likely to have central nervous system complications (P = .0358 and P = .023, respectively). Although no association was observed between an elevated MIC to fluconazole and mortality, those who received voriconazole or high-dose fluconazole (≥800 mg) for maintenance therapy were more likely to survive (P = .0288). Conclusions. Additional studies are required to further investigate the morbidity and mortality associated with an elevated MIC to fluconazole in cryptococcal meningitis, to determine when it is appropriate to perform susceptibility testing, and to evaluate its cost effectiveness.

Background The Xpert MTB/RIF (Xpert) assay is a rapid nucleic acid amplification test widely used in settings of high tuberculosis prevalence to detect tuberculosis as well asrpoBmutations associated with rifampin resistance. Data are needed on the diagnostic performance of Xpert in lower-prevalence settings to inform appropriate use for both tuberculosis detection and the need for respiratory isolation. Methods Xpert was compared to 2 sputum samples, each evaluated with acid-fast bacilli (AFB) smear and mycobacterial culture using liquid and solid culture media, from participants with suspected pulmonary tuberculosis from the United States, Brazil, and South Africa. Results Of 992 participants enrolled with evaluable results, 22% had culture-confirmed tuberculosis. In 638 (64%) US participants, 1 Xpert result demonstrated sensitivity of 85.2% (96.7% in participants with AFB smear-positive [AFB(+)] sputum, 59.3% with AFB smear-negative [AFB(-)] sputum), specificity of 99.2%, negative predictive value (NPV) of 97.6%, and positive predictive value of 94.9%. Results did not differ between higher- and low-prevalence settings. A second Xpert assay increased overall sensitivity to 91.1% (100% if AFB(+), 71.4% if AFB(-)), with specificity of 98.9%. In US participants, a single negative Xpert result predicted the absence of AFB(+)/culture-positive tuberculosis with an NPV of 99.7%; NPV of 2 Xpert assays was 100%, suggesting a role in removing patients from airborne infection isolation. Xpert detected tuberculosis DNA and mutations associated with rifampin resistance in 5 of 7 participants with rifampin-resistant, culture-positive tuberculosis. Specificity for rifampin resistance was 99.5% and NPV was 98.9%. Conclusions In the United States, Xpert testing performed comparably to 2 higher-tuberculosis-prevalence settings. These data support the use of Xpert in the initial evaluation of tuberculosis suspects and in algorithms assessing need for respiratory isolation.

Sex steroids are important regulators for longitudinal growth, bone mass accrual, and sexual dimorphism of the skeleton. 17β-Estradiol regulates proliferation and differentiation of female chondrocytes via a membrane-associated signaling pathway in addition to its estrogen receptor (ER) mediated effects. In contrast, testosterone does not elicit a similar membrane response, either in male or female cells. Whereas female rat growth plate chondrocytes convert testosterone to 17β-estradiol, male chondrocytes produce 5α-dihydrotestosterone (DHT). Previously DHT was found to mediate sex-specific effects of testosterone in male cells, but it is not known if a membrane-signaling pathway is involved. In this study, we hypothesized that DHT can induce sex-specific rapid membrane effects similar to other steroid hormones. Confluent cultures of chondrocytes isolated from resting zones of growth plates of both male and female rats were treated with 10(-10)-10(-7)M testosterone or DHT for 3, 9, 90 and 270min and protein kinase C (PKC) and phospholipase A2 (PLA2) activities were measured. To examine potential signaling pathways involved in PKC activation, male chondrocytes were treated with 10(-7)M DHT for 9min in the presence or absence of the phospholipase C (PLC) inhibitor U73122, the secretory PLA2 inhibitor quinacrine or the cytosolic PLA2 inhibitor AACOCF3; the Gαi inhibitor pertussis toxin (PTX) or Gαs activator cholera toxin (CTX), and the general G-protein inhibitor GDPβS; thapsigargin, an inhibitor of a Ca-ATPase pump in the endoplasmic reticulum; verapamil and nifedipine, inhibitors of specific L type Ca2+ channels on the cell membrane; and cyproterone acetate (CPA), which is an inhibitor of the classical androgen receptor (AR); as well as the transcription inhibitor actinomycin D, or the translation inhibitor cycloheximide. DHT induced a dose-dependent increase in PKC and PLA2 activity in male cells with the highest increase at 10(-7)M DHT (p

We sought to define the prevalence of blaZ gene types and the inoculum effect to cefazolin among methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream infections. The blaZ gene was present in 142/185 (77%) isolates. A total of 50 (27%) isolates had a ≥4-fold increase in the cefazolin MIC from a standard to a high inoculum, and 8 (4%) demonstrated a nonsusceptible cefazolin MIC, all type A blaZ strains. The efficacy of cefazolin in the presence of the inoculum effect requires further study.

Mesenchymal stem cells (MSCs) are regarded as a potential autologous source for cartilage repair, because they can differentiate into chondrocytes by transforming growth factor-beta (TGF-β) treatment under the 3-dimensional (3-D) culture condition. In addition to these molecular and biochemical methods, the mechanical regulation of differentiation and matrix formation by MSCs is only starting to be considered. Recently, mechanical loading has been shown to induce chondrogenesis of MSCs in vitro. In this study, we investigated the effects of a calibrated agitation on the chondrogenesis of human bone MSCs (MSCs) in a 3-D alginate culture (day 28) and on the maintenance of chondrogenic phenotypes. Biomechanical stimulation of MSCs increased: (i) types 1 and 2 collagen formation; (ii) the expression of chondrogenic markers such as COMP and SOX9; and (iii) the capacity to maintain the chondrogenic phenotypes. Notably, these effects were shown without TGF-β treatment. These results suggest that a mechanical stimulation could be an efficient method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering in a 3-D environment. Additionally, it appears that MSCs and chondrocyte responses to mechanical stimulation are not identical.

In 2010 the Clinical and Laboratory Standards Institute (CLSI) lowered the susceptibility breakpoints of some cephalosporins and aztreonam for Enterobacteriaceae and eliminated the need to perform screening for extended-spectrum β-lactamases (ESBLs) and confirmatory tests. The aim of this study was to determine how many ESBL-producing strains of three common species of Enterobacteriaceae test susceptible using the new breakpoints. As determined with the CLSI screening and confirmatory tests, 382 consecutive ESBL-producing strains were collected at Huashan Hospital between 2007 and 2008, including 158 strains of Escherichia coli , 164 of Klebsiella pneumoniae , and 60 of Proteus mirabilis . Susceptibility was determined by the CLSI agar dilution method. CTX-M-, TEM-, and SHV-specific genes were determined by PCR amplification and sequencing. bla CTX-M genes alone or in combination with bla SHV were present in 92.7% (354/382) of these ESBL-producing strains. Forty-two (25.6%) strains of K. pneumoniae harbored SHV-type ESBLs alone or in combination. No TEM ESBLs were found. Utilizing the new breakpoints, all 382 strains were resistant to cefazolin, cefotaxime, and ceftriaxone, while 85.0 to 96.7% of P. mirabilis strains tested susceptible to ceftazidime, cefepime, and aztreonam, 41.8 to 45.6% of E. coli strains appeared to be susceptible to ceftazidime and cefepime, and 20.1% of K. pneumoniae were susceptible to cefepime. In conclusion, all ESBL-producing strains of Enterobacteriaceae would be reported to be resistant to cefazolin, cefotaxime, and ceftriaxone by using the new CLSI breakpoints, but a substantial number of ESBL-containing P. mirabilis and E. coli strains would be reported to be susceptible to ceftazidime, cefepime, and aztreonam, which is likely due to the high prevalence of CTX-M type ESBLs.

Bone bridges linking the epiphysis and metaphysis termed “tethers” have been found in the femoral growth plates of C57Bl/6 mice and are disrupted when the vitamin D receptor (VDR) is ablated. It is unknown if tethers are found in other growth centers, if they are regulated in a comparable manner, or if they have a functional role in skeletal development or stability. To address this, distal femoral growth plates (GPs) and spheno-occipital synchondroses (SOSs) of wild-type C57Bl/6 mice from 2 to 15 weeks of age were analyzed using μCT scans. The GPs and SOSs of VDR+/+ and VDR−/− mice fed regular or rescue diets to restore mineral homeostasis until 10 weeks of age were also scanned. Tethers in GPs and SOSs both thickened and accumulated in number as these growth centers decreased in size. Ablating the VDR made GPs and SOSs rachitic and nearly eliminated tether formation. Rescue diets restored the volume of both growth centers but only partially restored growth center thickness and tether formation, suggesting that lα,25-dihydroxy vitamin D3 partially regulates tether formation in these growth centers via its receptor. In VDR+/+ mice 2–15 weeks in age, growth center thickness was inversely correlated to animal weight whereas tether phenotype (tether volume/growth center volume, tether number/mm, tether width, tether spacing) was significantly related to animal weight. In both 2–15 week old VDR+/+ and 10 week old VDR+/+ and VDR−/− mice on normal and rescue diets, tether phenotype (tether number/mm, tether spacing) had strikingly similar relationships to growth center thickness. These results show that tethers are present in growth centers in different anatomic and undergo developmental changes in a comparable manner; in both sites, VDR-regulated tether formation is strongly linked to growth center morphology; and tether formation is associated with body weight, suggesting a role in maintaining growth plate stability during growth.

Histoplasmosis is known to be an AIDS-associated infection, with scattered areas of endemicity throughout the world. Although the Atlanta, GA, metropolitan area is not a highly endemic area, a significant number of cases have been noted at our institution in recent years. Cases of histoplasmosis over a 4-year period were reviewed. All 27 patients (100%) were HIV infected. Thirty percent of patients with histoplasmosis were from Latin American countries. Patients from Latin America were younger than patients from the United States, tended to be more likely to have proven disease, and were exclusively male. Patients with proven disease had significantly higher urine histoplasma antigen levels, lower platelets counts, and lower neutrophil counts than patients with probable disease. The majority of patients survived after treatment with antifungals and initiation of antiretroviral therapy. Histoplasmosis is thus an important consideration in the workup of patients with advanced HIV in nonendemic areas of the United States.

The aim of this study was to establish a critical-sized nonjoint chondral defect animal model and to evaluate its feasibility for testing cartilage regeneration strategies. Dermal biopsy punches 1-4 mm in diameter were used to create cylindrical full-thickness defects in the center of athymic rat xiphoids. The 3 and 4 mm defects remained unhealed 35 days postsurgery, with a large area in the center that had low proteoglycan content based on contrast-enhanced microCT (EPIC-microCT), radiographic, and histological analyses. In a second step, tissue-engineered cartilage was synthesized by culturing primary bovine articular chondrocytes on poly-L-lactic acid (PLA) scaffolds in a perfusion-shear bioreactor for 28 days. These chondrocyte/PLA constructs or primary bovine chondrocytes were implanted into 3-mm-diameter defects. Empty defects and defects implanted with empty PLA scaffolds were used as controls. Xiphoids were harvested 28 days after surgery and examined with faxitron, microCT, and histology using hematoxylin and eosin and safranin-O staining. Both chondrocyte/PLA constructs and chondrocytes alone formed neocartilage. The results indicate that a 3 mm cylindrical defect in a rat xiphoid is an economic, feasible, and reproductive model to evaluate the potential of various constructs for nonjoint cartilage repair.

Herpes simplex virus (HSV) encephalitis has a predilection for the temporal and frontal lobes but occasionally affects the brainstem. We describe a patient who developed HSV brainstem encephalitis that progressed to quadriplegia. Using MEDLINE, we conducted a comprehensive review of other published cases of HSV brainstem encephalitis. Twenty-four published cases of HSV brainstem encephalitis met our inclusion criteria. The mean age was 41.4 years (range 18-71). HSV-1 was the etiologic agent in 79% of reported HSV brainstem encephalitis cases, and HSV-2 accounted for 21% of cases. Infection was limited to the brainstem in 29% of cases and multi-focal, including the brainstem, in 71%. Common manifestations of HSV brainstem encephalitis included neuro-ophthalmologic findings (81%), cranial nerve deficits (69%), and fever (69%). Quadriplegia, as occurred in our patient, was an unusual finding (19%). The mortality rate of HSV brainstem encephalitis was 41%. Intravenous acyclovir showed a beneficial effect on mortality (75% vs. 22%, p = 0.06). HSV brainstem encephalitis is a distinct type of HSV encephalitis. With the increasing use of HSV-PCR, more cases of HSV brainstem encephalitis may be identified. A greater recognition of this syndrome will help better define its optimal treatment and prognosis.

Coagulase-negative staphylococcal species are generally considered commensal inhabitants of the skin. Often regarded as avirulent organisms, certain species such as Staphylococcus lugdunensis have been identified as the cause of severe human infections including infective endocarditis. We report on a case of a native valve, S. lugdunensis infective endocarditis relapse after initial conservative medical therapy. A literature review of the outcomes of treatment was also performed. Analyzing definitive medical versus medical-surgical treatment of native valve infective endocarditis, an odds ratio of mortality of 3.1875 (95% confidence interval [CI], 1.1855-8.5705; P < 0.03) was obtained and was significant based on 2-tailed Fisher exact probability test. This analysis reveals an increase risk of mortality with medical therapy alone versus medical-surgical therapy for native valve infective endocarditis. Our case and review of the literature emphasize the growing evidence that early medical treatment and surgical evaluation and removal of infected foreign hardware is required to prevent significant morbidity and mortality.

Background: Sexually transmissible infections (STI) have been variably associated with increased risks of prostate cancer, largely in case-control studies. Methods: In the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, we examined risk of prostate cancer in relation to serum antibodies to Chlamydia trachomatis, human papillomavirus-16 and -18, herpes simplex virus-2, cytomegalovirus, and human herpesvirus-8 in 868 cases (765 Whites and 103 Blacks) and 1,283 controls matched by race, age, time since initial screening, and year of blood draw; all blood samples were collected at least 1 year before prostate cancer diagnosis, except for 43 Black cases. We also assessed risk associated with self-reported history of syphilis and gonorrhea. Results: Prevalences of the 7 STIs among controls were weakly correlated, and all were more frequent among Blacks than Whites, except for human herpesvirus-8. Among Whites, prostate cancer risk was not significantly associated with the individual infections or with their number (Ptrend = 0.1); however, men with one or more STI had slightly higher risk (odds ratio, 1.3; 95% confidence interval, 1.0-1.6). Among Blacks, excess risk was associated with IgA antibody to C. trachomatis (odds ratio, 2.1; 95% confidence interval, 1.2-3.6). Conclusion: This large prospective study of prostate cancer shows no consistent association with specific STIs and a borderline association with any versus none. Whether a shared response or correlated infection not directly measured underlies the weak association requires further study. (Cancer Epidemiol Biomarkers Prev 2008;17(9):2374–81)

To investigate whether the chondrocytes-alginate construct properties, such as cell seeding density and alginate concentration might affect the redifferentiation, dedifferentiated rat articular chondrocytes were encapsulated at low density (LD: 3 x 10(6) cells/ml) or high density (HD: 10 x 10(6) cells/ml) in two different concentrations of alginate gel (1.2% or 2%, w/v) to induce redifferentiation. Cell viability and cell proliferation of LD culture was higher than those of HD culture. The increase in alginate gel concentration did not make an obvious difference in cell viability, but reduced cell proliferation rate accompanied with the decrease of cell population in S phase and G2/M phase. Scan electron microscopy observation revealed that chondrocytes maintained round in shape and several direct cell-cell contacts were noted in HD culture. In addition, more extracellular matrix was observed in the pericellular region of chondrocytes in 2% alginate culture than those in 1.2% alginate culture. The same tendency was found for the synthesis of collagen type II. No noticeable expression of collagen type I was detected in all constructs at the end of 28-day cultures. These results suggested that construct properties play an important role in the process of chondrocytes' redifferentiation and should be considered for creating of an appropriate engineered articular cartilage.

Both chondrocytes and mensenchymal stem cells (MSCs) are the most used cell sources for cartilage tissue engineering. However, monolayer expansion to obtain sufficient cells leads to a rapid chondrocyte dedifferentiation and a subsequent ancillary reduced ability of MSCs to differentiate into chondrocytes, thus limiting their application in cartilage repair. The aim of this study was to investigate the influence of the monolayer expansion on the immunophenotype and the gene expression profile of both cell types, and to find the appropriate compromise between monolayer expansion and the remaining chondrogenic characteristics. To this end, human chondrocytes, isolated enzymatically from femoral head slice, and human MSCs, derived from bone marrow, were maintained in monolayer culture up to passage 5. The respective expressions of cell surface markers (CD34, CD45, CD73, CD90, CD105, CD166) and several chondrogenic-related genes for each passage (P0-P5) of those cells were then analyzed using flow cytometry and quantitative real-time PCR, respectively. Flow cytometry analyses showed that, during the monolayer expansion, some qualitative and quantitative regulations occur for the expression of cell surface markers. A rapid increase in mRNA expression of type 1 collagen occurs whereas a significant decrease of type 2 collagen and Sox 9 was observed in chondrocytes through the successive passages. On the other hand, the expansion did not induced obvious change in MSCs gene expression. In conclusion, our results suggest that passage 1 might be the up-limit for chondrocytes in order to achieve their subsequent redifferentiation in 3D scaffold. Nevertheless, MSCs could be expanded in monolayer until passage 5 without loosing their undifferentiated phenotypes.

Pluripotent stem cell aggregates offer an attractive approach to emulate embryonic morphogenesis and skeletal development. Calcium phosphate (CaP) based biomaterials have been shown to promote bone healing due to their osteoconductive and potential osteoinductive properties. In this study, we hypothesized that incorporation of CaP-coated hydroxyapatite mineral particles (MPs) within murine embryonic stem cell (ESC) aggregates could promote osteo-chondrogenic differentiation. Our results demonstrated that MP alone dose-dependently promoted the gene expression of chondrogenic and early osteogenic markers. In combination with soluble osteoinductive cues, MPs enhanced the hypertrophic and osteogenic phenotype, and mineralization of ESC aggregates. Additionally, MPs dose-dependently reduced ESC pluripotency and thereby decreased the size of teratomas derived from MP-incorporated ESC aggregates in vivo . Our data suggested a novel yet simple means of using mineral particles to control stem cell fate and create an osteochondral niche for skeletal tissue engineering applications. Statement of Significance Directing stem cell differentiation and morphogenesis via biomaterials represents a novel strategy to promote cell fates and tissue formation. Our study demonstrates the ability of calcium phosphate-based mineral particles to promote osteochondrogenic differentiation of embryonic stem cell aggregates as well as modulate teratoma formation in vivo . This hybrid biomaterial–ESC aggregate approach serves as an enabling platform to evaluate the ability of biomaterials to regulate stem cell fate and regenerate functional skeletal tissues for clinical applications.

Young pregnant women are a group at particularly high risk of STDs.1,2 In a systematic literature review of sexual risk and STDs among pregnant teenagers and those who have given birth, STD incidence was 19−39% during pregnancy, and 14−39% between six and 10 months postpartum. In addition, adolescent mothers were twice as likely as their nulliparous peers to acquire an STD during a 12-month period. Furthermore, 78−88% of pregnant women engaged in sex without condoms during pregnancy, and pregnant women were only about one-fifth as likely as nonpregnant women to use condoms.3 Current guidelines from the Centers for Disease Control and Prevention (CDC) recommend STD screening for all high-risk women-usually defined as those younger than 25 and those with multiple or new partners.4 Frequently, screening for all high-risk pregnant women occurs only during initial prenatal care visits, so an infection that is acquired later in pregnancy may go undetected. Some clinics retest women who are deemed high-risk (e.g., those testing STD-positive at their initial visit) or exhibit symptoms; however, testing procedures differ from clinic to clinic, and screening behavior can vary by provider.5 In addition, because an STD diagnosis and treatment are not enough to have long-term effects on risky behavior,6 an emphasis on prevention is necessary to achieve long-term risk reduction. Prevention programs integrated with prenatal care can capitalize on women's motivation to have a healthy pregnancy and child. HIV and other STD prevention programs have been successfully integrated in psychiatric care, drug treatment and palliative medicine settings.7–9 This model for prevention could be extended to include prenatal care. For interventions to be most effective in clinical settings, they should be easy to implement and tailored to meet the needs and risks of particular patients. Thus, it may be beneficial to identify subgroups of women at varying risk of STDs during pregnancy, and implement treatment, care and prevention programs that best meet the needs of those individuals. To successfully do this, providers need to go beyond the typical characteristics obtained during prenatal care assessments. A 2002 Institute of Medicine report and more recent research syntheses suggest that ecological approaches to health, and to STD prevention, are particularly useful and needed.3,10,11 Ecological systems theory emphasizes that factors from many levels (e.g., individual, dyad, family and community) can influence health.3,10–12 The few studies on young people's sexual activity that have included factors from many levels have shown that dyad, family and community factors independently contribute to risk behavior above and beyond individual factors.13–15 In addition to using primarily individual-level predictors, most studies that have tried to estimate STD risk have used standard analytic techniques (e.g., logistic regression). However, these methods are limited because they assume primarily linear relationships between the predictors and outcomes that may not be realistic in applied settings. Furthermore, results from these techniques (e.g., odds ratios in logistic regression) are difficult to translate into clinical practice and policy.16 Classification tree analysis, also called recursive partitioning, avoids the limitations associated with logistic regression and other standard methods. The technique uses a set of predictors to create subgroups of individuals that vary in risk for the main outcome of interest, ultimately producing a user-friendly classification tree that can be employed to guide care, treatment and prevention efforts in clinical settings. For example, in a study of young adults at an STD clinic, several subgroups were classified as having a high STD risk because of particular behavioral and emotional factors (e.g., one group felt bad about themselves after having sex and had sex to relieve tension; one group fell bad about themselves after having sex and had sex to get back at someone).17 Use of classification tree analysis has been documented in the health care and managed care literature,18 and has been used in many clinical and medical settings to identify high-risk patients and to help create clinical guidelines.19–23 Clinicians often use informal decision trees when making clinical decisions. On the basis of a complex interaction of medical, demographic and behavioral factors, clinicians determine the likelihood of disease outcomes and often use these patient characteristics to guide care and treatment decisions (e.g., if a woman is having risky sex and is younger than 25, the clinician may screen for STDs because past experience suggests that the patient is at risk). The purpose of this study is to use individual, dyad, and family- and community-level characteristics to develop a clinically relevant classification tree that will help identify women who are at risk of acquiring an STD during pregnancy and will provide a formal decision-making structure for STD prevention, treatment and care.

The progressive loss of endogenous regenerative capacity that accompanies mammalian aging has been attributed at least in part to alterations in the extracellular matrix (ECM) composition of adult tissues. Thus, creation of a more regenerative microenvironment, analogous to embryonic morphogenesis, may be achieved via pluripotent embryonic stem cell (ESC) differentiation and derivation of devitalized materials as an alternative to decellularized adult tissues, such as demineralized bone matrix (DBM). Transplantation of devitalized ESC materials represents a novel approach to promote functional tissue regeneration and reduce the inherent batch-to-batch variability of allograft-derived materials. In this study, the osteoinductivity of embryoid body-derived material (EBM) was compared to DBM in a standard in vivo ectopic osteoinduction assay in nude mice. EBM derived from EBs differentiated for 10 days with osteogenic media (+β-glycerophosphate) exhibited similar osteoinductivity to active DBM (osteoinduction score = 2.50 ± 0.27 vs. 2.75 ± 0.16) based on histological scoring and exceeded inactive DBM (1.13 ± 0.13, p de novo bone formation in vivo.

Background Whether community-associated methicillin-resistant Staphylococcus aureus (MRSA) genotypes (e.g., USA300) are a major cause of bloodstream infections (BSIs) and health care-associated infections has been poorly defined. Methods Consecutive MRSA isolates recovered from patients with BSIs were prospectively collected at an urban public hospital. Molecular typing studies were performed. Prevalence and risk factors for the MRSA USA300 genotype were assessed. Results One hundred thirty-two cases of MRSA BSI were documented over 7.5 months in 2004 (incidence, 6.79 per 1000 admissions); 116 isolates were available for genotyping. Characteristics of the 116 evaluable cases included: a mean age 47 years; 62% were male, 82% were African American, and 22% were HIV seropositive. The crude in-hospital mortality rate was 22%. In 107 cases (92%), there was contact with a health care facility within the year prior to infection, and a nosocomial infection (defined as positive blood culture results obtained >48 h after admission) occurred in 49 cases (42%). PFGE demonstrated that 39 (34%) of the 116 isolates were the MRSA USA300 genotype; 34 (29%) were USA100; 42 (36%) were USA500; and 1 (1%) was USA800. MRSA USA300 accounted for 28% of health care-associated BSIs and 20% of nosocomial MRSA BSIs. In multivariate analysis, isolation of the USA300 genotype was associated with injectiondrug use (OR, 3.67; 95% CI, 1.10-12.28) and skin and soft tissue infection (OR, 4.26; 95% CI, 1.08-16.84). Patients who resided in long-term care facilities (OR, 0.09; 95% CI, 0.01-0.82) and those who were treated with antimicrobials in the prior year were less likely to have MRSA USA300 genotype recovered (OR, 0.10; 95% CI, 0.02-0.49). Conclusions MRSA USA300 genotype, the predominant cause of community-associated MRSA infections in our area (Atlanta, GA), has now emerged as a significant cause of health care-associated and nosocomial BSI. MRSA USA300 as a nosocomial pathogen presents new challenges to infection control programs.

To the Editor: Multidrug-resistant tuberculosis (MDR TB), i.e., TB resistant to at least the 2 most effective first-line antituberculous drugs (isoniazid [INH] and rifampin [RIF]), is increasing globally. World Health Organization estimations of 390,000–510,000 new MDR TB cases and 150,000 related deaths in 2008 highlight the need for timely drug susceptibility testing and improved therapies (1). Although novel rapid drug susceptibility testing tools are increasingly available, their clinical applicability is unsettled. We report a patient with pulmonary TB relapse with discordant genotypic and in vitro phenotypic drug susceptibility testing results associated with a mutation outside the RIF resistance determining region (RRDR) of the rpoB gene. In August 2009, a 45-year-old homeless woman with AIDS (CD4+ T-cell count 3 cells/mm3) and a history of substance abuse sought care for fever, night sweats, weight loss, and cough (Table A1). Pulmonary TB had been diagnosed in June 2008. At that time, she received, by directly observed therapy, 6 weeks of INH, RIF, pyrazinamide (PZA), and ethambutol (EMB) through the local health department and was switched to RIF, PZA, and EMB on week 7 after the isolate was determined by liquid culture with BD BACTEC MGIT 960 Mycobacterial Testing System (BD Diagnostics, Sparks, MD, USA) to be INH resistant. During that period, she resided in American Lung Association–supported housing for TB patients and had 97% medication adherence by dose count. Her condition clinically improved, infiltrates completely resolved according to chest radiograph, 12 sputum inductions failed to yield sufficient material for analysis, and she began highly active antiretroviral therapy (HAART). In December 2008, however, because of crack cocaine use and belligerent behavior, she lost housing privileges. Caseworkers could not locate her to complete the 9-month planned directly observed therapy. The woman was hospitalized in January and again in February 2009 with dyspnea and off medications. In both instances, chest radiographs showed no new changes, sputum specimens were negative for acid-fast bacilli (AFB) by microscopy and culture, and she was treated for presumptive Pneumocystis pneumonia and showed clinical improvement. In August 2009, she was readmitted with cough and new cavitation on chest radiograph. Chest computed tomographic scan demonstrated right upper lobe infiltrates, bilateral lower lobe cavitation, and hilar and mediastinal lymphadenopathy. Sputum AFB smear was positive (graded 4+), and nucleic acid amplification (Amplified Mycobacterium Direct Test; Gen-Probe, San Diego, CA, USA) was positive for Mycobacterium tuberculosis complex. INH, RIF, PZA, and EMB, along with moxifloxacin (MXF) and amikacin (AMK), were initiated in accordance with 2003 national TB treatment guidelines for possible MDR TB (2). Shortly thereafter, a line probe assay (GenoType MTBDRplus; HAIN Lifescience, Nehren, Germany) performed by Southeastern National TB Center (Gainesville, FL, USA) on the culture of the sputum specimen obtained at admission indicated an inhA point mutation but no mutation in the RRDR region of the rpoB gene, which suggested that the isolate was INH resistant but RIF susceptible. AMK was discontinued, and the patient was discharged with RIF, PZA, EMB, and MXF. One week later, drug susceptibility testing (BD BACTEC MGIT 960 System) results from the state mycobacteriology laboratory demonstrated that the M. tuberculosis isolate was resistant to INH and RIF. The patient was readmitted to resume injectable aminoglycoside therapy. After 5 weeks, sputum culture became negative, clinical and radiographic improvement was apparent, and HAART was reinitiated. She completed 2 months’ INH/PZA/EMB/MXF/AMK inpatient therapy and was discharged to complete 6 additional months of PZA/EMB/MXF and streptomycin followed by 16 months of PZA/EMB/MFX. With HAART, her plasma HIV RNA viral load became undetectable, but her CD4 count remained low (9 cells/mm3). She died from a motor vehicle accident 10 months after recurrent TB was diagnosed. In this patient, RIF resistance was not predicted by line probe assay but was identified phenotypically by an automated system (BD BACTEC MGIT 960 System) that continuously monitors for growth and detection of mycobacteria. Through genotyping and DNA sequencing of the 2008 and 2009 M. tuberculosis isolates, the Mycobacteriology Laboratory Branch at the Centers for Disease Control and Prevention (Atlanta, GA, USA) established that the 2009 infection was a relapse, not re-infection, and confirmed the inhA mutation in both isolates. Using primers extending beyond the RRDR (the rpoB region surveyed by rapid molecular tests and responsible for >95% of RIF resistance mutations) the laboratory identified a novel rpoB gene mutation at codon 480 (ACC→AAC; Thr→Asn) and another previously described (3–5) mutation at codon 176 (GTC→TTC; Val→Phe) in the 2009 isolate, which has been implicated in RIF resistance. The role of the T480N mutation in RIF resistance is being investigated. This case demonstrates the limitations of rapid molecular drug susceptibility testing (6). Rapid molecular diagnostics are valuable adjuncts to conventional phenotypic testing because they can quickly confirm clinically suspected MDR TB and have high agreement with other genotypic and phenotypic methods (7–10). However, they should not supplant phenotypic testing, and clinicians should understand their limitations. When rapid molecular tests are negative but suspicion for MDR TB is high, MDR TB treatment should be continued until phenotypic susceptibility results are available. DNA sequencing may be best suited for evaluating suspected drug-resistant M. tuberculosis isolates with discordant results for phenotypic susceptibility and rapid molecular testing.

Introduction The Centers for Disease Control and Prevention has recommended using a nucleic acid amplification test (NAAT) for diagnosing pulmonary tuberculosis (TB) but there is a lack of data on NAAT cost-effectiveness. Methods We conducted a prospective cohort study that included all patients with an AFB smear-positive respiratory specimen at Grady Memorial Hospital in Atlanta, GA, USA between January 2002 and June 2008. We determined the sensitivity, specificity, and positive and negative predictive value of a commercially available and FDA-approved NAAT (amplified MTD, Gen-Probe) compared to the gold standard of culture. A cost analysis was performed and included costs related to laboratory tests, hospital charges, anti-TB medications, and contact investigations. Average cost per patient was calculated under two conditions: (1) using a NAAT on all AFB smear-postive respiratory specimens and (2) not using a NAAT. One-way sensitivity analyses were conducted to determine sensitivity of cost difference to reasonable ranges of model inputs. Results During a 6 1/2 year study period, there were 1,009 patients with an AFB smear-positive respiratory specimen at our public urban hospital. We found the NAAT to be highly sensitive (99.6%) and specific (99.1%) on AFB smear-positive specimens compared to culture. Overall, the positive predictive value (PPV) of an AFB smear-positive respiratory specimen for culture-confirmed TB was 27%. The PPV of an AFB smear-positive respiratory specimen for culture-confirmed TB was significantly higher for HIV-uninfected persons compared to those who were HIV-seropositive (152/271 [56%] vs. 85/445 [19%]; RR = 2.94, 95% CI 2.36–3.65, p

Objectives Nosocomial pathogens such as Acinetobacter baumannii are a growing public health threat, due in part to their increasing resistance to antibiotics. Since some strains are resistant to all available antibiotics, novel therapies are urgently needed. Plasmablasts are short-lived B cells found in the blood that can be collected and harnessed to produce therapeutic antibodies. We set out to determine whether plasmablasts are induced during infection with A. baumannii and other nosocomial pathogens. Methods We obtained blood samples from patients infected with antibiotic-resistant nosocomial pathogens, and analysed their plasmablast response by flow cytometry. Results We observed a strong induction of plasmablasts in patients with antibiotic-resistant A. baumannii infection. Furthermore, plasmablasts were also induced in response to other drug-resistant nosocomial pathogens. Conclusions These data suggest that plasmablasts may be broadly harnessed to develop therapeutic antibodies to combat otherwise untreatable antibiotic-resistant infections.

1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] is crucial for normal skeletal development and bone homeostasis. Protein disulfide isomerase family A, member 3 (PDIA3) mediates 1α,25(OH)2D3 initiated-rapid membrane signaling in several cell types. To understand its role in regulating skeletal development, we generated Pdia3-deficient mice and examined the physiologic consequence of Pdia3-disruption in embryos and Pdia3 +/− heterozygotes at different ages. No mice homozygous for the Pdia3-deletion were found at birth nor were there embryos after E12.5, indicating that targeted disruption of the Pdia3 gene resulted in early embryonic lethality. Pdia3-deficiency also resulted in skeletal manifestations as revealed by µCT analysis of the tibias. In comparison to wild type mice, Pdia3 heterozygous mice displayed expanded growth plates associated with decreased tether formation. Histomorphometry also showed that the hypertrophic zone in Pdia3 +/− mice was more cellular than seen in wild type growth plates. Metaphyseal trabecular bone in Pdia3 +/− mice exhibited an age-dependent phenotype with lower BV/TV and trabecular numbers, which was most pronounced at 15 weeks of age. Bone marrow cells from Pdia3 +/− mice exhibited impaired osteoblastic differentiation, based on reduced expression of osteoblast markers and mineral deposition compared to cells from wild type animals. Collectively, our findings provide in vivo evidence that PDIA3 is essential for normal skeletal development. The fact that the Pdia3 +/− heterozygous mice share a similar growth plate and bone phenotype to nVdr knockout mice, suggests that PDIA3-mediated rapid membrane signaling might be an alternative mechanism responsible for 1α,25(OH)2D3’s actions in regulating skeletal development.